PROPOSAL SUMMARY (ABSTRACT) Many pathologies in numerous organ systems are associated with fibrosis. Fibrosis, characterized by tissue stiffening, occurs when cells are unable to maintain the mechanical properties of tissues, determined primarily by their extracellular matrix (ECM). In healthy tissues, cells maintain ECM composition and organization through feedback signaling that can sense the stiffness of the physical microenvironment and maintain the mechanical steady state. Dysfunction of this feedback signaling in cells can lead to an imbalance between synthesis and degradation of matrix resulting in tissue stiffening, fibrosis, and tissue malfunction. Unfortunately, the molecular mechanisms that control mechanical homeostasis remain largely unknown. This lack of knowledge constitutes a major limitation for understanding fibrosis and developing possible treatments. The proposed study will address this gap in knowledge by investigating novel mechanisms of mechanical homeostasis in vitro and in vivo. One key process in the maintenance of mechanical homeostasis is the cells ability to sense and respond appropriately to the stiffness of the surrounding ECM. Cells bind directly to the ECM through focal adhesions (FAs). FAs are intracellular protein complexes at the cell membrane-ECM interface that mediate ECM-dependent signaling pathways. FAs mediate cell response to the ECM by changing composition and organization of over 200 proteins. However, it is currently unknown what mechanism(s) enable this dynamic regulation of proteins at FA complexes in response ECM stiffness. My preliminary data identified local translation of FA proteins occurring directly at the FAs. Additionally, I identified mRNA regulatory factors including miRNAs and RNA binding proteins localized at FAs. Thus, the goal of this proposal is to determine if regulation of local translation mediates cell- ECM stiffness interactions. Using a cell culture system (in vitro), I will pinpoint the specific regulatory factors that mediate local translation and determine their role in the response to changes in substrate stiffness (Aim 1). Fibrosis occurs in 3D tissues which harbor a complex physical microenvironment. Therefore, I will use the zebrafish fin-fold regeneration model (in vivo) to visualize and disrupt mRNA localization to FAs during tissue regeneration (Aim 2). Together, these proposed experiments will fill a critical gap in knowledge for the study of cell-ECM interactions and, therefore, mechanical homeostasis. Discovering the mechanism(s) of physiological matrix homeostasis are critical for overcoming current barriers in understanding fibrosis and developing effective treatments.